Chemists
at The Scripps Research Institute (TSRI) have found an easier way to perform
one of the most fundamental tasks in molecular biology. Their new method allows
scientists to add a marker to certain cells, so that these cells may be easily
located and/or selected out from a larger cell population.

The technique,
which is described in a recent issue of the chemistry journal Angewandte Chemie International Edition,
makes use of the tight binding of two proteins that are cheaply obtainable but
are not found in human or other mammalian cells. As such, it has advantages
over existing cell-marking techniques.

“This
new technique is cheap, easy and sensitive,” said TSRI Institute Professor
Richard A. Lerner, who is the senior author of the new report. “The method
should be useful in a variety of applications that require separating out
certain types of cells.”

Looking for a Better Way

The
best-known cell marker in use today is GFP (green fluorescent protein), a
jellyfish protein that emits a distinctive green light when illuminated by
certain other light wavelengths. When scientists want to add a new gene to
cells, for example to produce a therapeutic protein, they often construct a
genetic sequence that also includes the GFP gene. Thus the cells that
successfully produce the new protein will also produce GFP, whose fluorescence
allows these cells to be identified and even sorted out from a larger
population.

But
fluorescence-based cell sorting is relatively expensive and cumbersome.
Alternative cell-marking techniques use marker molecules to which antibodies or
metals will bind tightly, but these are apt to have unwanted side effects on
the cells that they mark. Lerner’s team, led by first author Yingjie Peng, a postdoctoral
fellow, set out to invent a better method.

The
new method exploits a special property of chitinase enzymes, which evolved to break
down chitin—a tough, sugar-derived material found, for example, in crab shells,
squid beaks and the cell walls of fungi. In addition to a main chitin-breaking
domain, chitinases have another active structure, a “chitin binding domain”
(ChBD). “It makes a super-strong bond with chitin,” said Peng. In recent years,
scientists have begun to use this high-affinity binding of ChBD and chitin as a
marker system, typically for selecting ChBD-tagged proteins in a lab dish. The new
method uses ChBD to mark and select cells.

A Powerful Tool

In the
basic technique, a new gene can be added to cells within a larger DNA vector
that also includes the genetic sequences for ChBD and GFP. The ChBD molecule
will be produced in such a way that it ends up being held on the outer surface
of its host cell’s plasma membrane—and the GFP molecule will sit just inside
the membrane. The GFP serves as a visual beacon, while the ChBD serves as a
handy gripping point for cell selection.

After
exposing a culture of test cells to this experimental ChBD-containing vector, the
scientists was able to see, via the GFP tags, which cells were expressing them,
and was able to select them out easily, with high sensitivity, using magnetic beads
coated with chitin. “This is a relatively easy benchtop method,” Peng said.
Importantly, these selected cells could produce progeny cells that seemed
normal and healthy.

Because
the ChBD marker, in the vector, is produced in a way that anchors it to a cell’s
membrane, it also can serve as a powerful tool for selecting just the membrane
fraction of a sample of cellular material. Peng and his colleagues demonstrated
this using chitin beads to quickly isolate a pure fraction of membrane material
from ChBD-marked test cells.

Cellulase
enzymes, which break down the ubiquitous plant compound cellulose, also have a
high-affinity cellulase-binding domain, which can be employed in the same way
as the ChBD.

The
scientists expect that the new cell-marking method will help to streamline
another major molecular biology technique, which was pioneered by the Lerner
laboratory in parallel with the group of Sir Gregory Winter at the Laboratory
of Molecular Biology in Britain. This technique allows scientists to produce
very large and diverse libraries of antibody arms, and to sift through them, or
“pan”—as gold miners pan for nuggets—for those that might be of use, for
example in therapies. ChBD-based markers should be useful in boosting the
efficiency of this panning process, said Peng.

The
Lerner laboratory is also investigating the potential use of ChBD-based cell
marking in living animals, for example to track the fates of selected cell
types throughout an animal’s lifespan.

The
research was supported by a grant from Scripps Korea Antibody Institute.

Send comments to: press[at]scripps.edu

“This
is a relatively easy benchtop method,” says Research Associate Yingjie Peng of
the Lerner lab. (Photo by Cindy Brauer.)

The
Lerner lab's simple and inexpensive method to mark cells uses vectors
that express the chitin-binding domain (ChBD) on eukaryotic cell
surfaces. The ChBD is linked to enhanced green fluorescent protein
(EGFP) through a protein that spans the plasma membrane.